One major outcome of the study of chemotaxis during the past decade is that the well-studied enteric model may not be representative of archaea and eubacteria ("bacteria'). Taxis in the archeon Halobacterium salinarum bears some remarkable similarities to that in Bacillus subtilis, a finding that suggests that the ancestral mechanism might have been closer to these mechanisms, rather than to the simplified mechanism in the enteric bacteria. For this reason, this work might establish a new paradigm for chemotaxis, which may be followed by the great sweep of eubacteria and archaea. It is becoming increasingly clear that complexes of receptors with other proteins control output, not just in chemotaxis but in many systems. However, bacterial chemotaxis is an especially good model system as virtually any combination of mutations, including the charge at each site of receptor methylation, can be used to ask specific questions and the effects studied in vivo and in vitro. In this proposal we are focusing on the interactions among chemotaxis proteins, including the receptors and the excitatory CheA kinase, in order to define the structure/function relationships. Our recent experiments have led to the hypothesis that CheY-P feeds back on the receptor to inhibit CheA during adaptation and may even be hydrolyzed at the receptor, unknown features in Escherichia coli chemotaxis. Such an interaction may occur in H. salinarum and may represent the ancestral adaptation mechanism. It appears that the receptors control CheY-P levels through differential regulation of the state of methylation/amidation of each of the sites of receptor methylation. Some proteins have no counterparts in E. coli -- CheC, CheD, and CheV. It appears that CheC/CheD, present also in H. salinarum, controls the processes that regulate CheY-P levels. Phosphorylation of CheV helps bring about adaptation. Exploring these subjects may revolutionize our understanding of how chemotaxis works in B. subtilis and perhaps in many bacteria. The goal is to elucidate these specific hypotheses or findings. Where the receptors and CheY-P interact and the consequences of the interaction in vivo and in vitro will be determined. The effect of CheV on this interaction will be explored. How degree of methylation at each site in the asparagine receptor, McpB, affects CheA and the CheY-P interaction as well as behavior will be quantitated. The purpose and location of interactions of CheC and CheD with each other and with McpB and CheA (in the case of CheC) will be elucidated. The (fast) kinetics of CheA autophosphorylation and phosphotransfer to CheY, CheB, and CheV and their purposes will be studied. The effect of receptor interaction on these kinetics will be explored.